This proposal aims to further develop a bioassay for the rapid determination of transporter affinity. We focus our proposed research on a pharmacologically relevant transport system, the small intestinal peptide transporter (PepT1). PepT1 plays a key role in intestinal absorption of di- and tripeptides, and mediates the absorption of a wide range of therapeutic compounds, such as aminopenicillins, cephalosporins, and 'angiotensin converting enzyme' (ACE) inhibitors. We have developed novel, selective, peptidomimetic fluorescent compounds with affinity for PEPT1 that can be used in high-throughput bioassays. An epithelial cell line stably expressing human PepT1 will be used to validate and optimize the assay. Successful implementation of this assay will significantly increase our understanding of the structural interactions that drive intestinal peptide transport and further our structural knowledge of SLC proteins in general. Additionally, it may aid future development of strategies to optimize the oral bioavailability of drugs by targeting specifically to PepT1 and the rational development of chemotherapeutics that are targeted to tumors overexpressing PepT1, such as pancreatic adenocarcinoma. Ou specific aims are: 1. To synthesize and evaluate novel fluorescent peptidomimetic substrates for PepT1In this aim we will design and synthesize novel fluorescent PepT1 substrates and select those that display a combination of high relative fluorescence quantum yield, metabolic stability in cell culture system, and high hPepTI affinity for further testing in a high-throughput setting. 2. Assay validation and optimization. In this specific aim we will validate our bioassay by determining assay accuracy, precision, limit of detection as well as limit of quantitation for known standards, specificity, linearity and range, and robustness. The long-term goal of this study is to develop a reliable, cost efficient, high-throughput bioassay for the peptide transporter system. This will aid in our understanding of the structural features that govern peptide and drug transport via this system and will significantly support studies aiming to target this transporter.